This grant application seeks to renew funding for an ongoing project designed to elucidate the mechanisms that underlie multiple functions of the glycoprotein (GP) Ib-IX-V receptor complex, specifically as they relate to the interaction with its main ligands, thrombin and von Willebrand factor (VWF). We propose to integrate structural information, ex vivo mutagenesis of specific protein domains and in vivo modification of cell expressed GPIb1 to establish how 1-thrombin binds to this receptor, how the interaction affects thrombin- mediated procoagulant and anticoagulant pathways, and the relationship with the binding of the VWF A1 domain (VWF-A1). These studies will demonstrate that GPIb is a key modulator of thrombin function during the response to vascular injury, and will highlight in this context a novel prothrombotic role of thrombin, independent of proteolytic activity and coordinated through concurrent interactions with VWF-A1 and GPIb. We will establish the structural determinants of this trimolecular assembly, investigate how it affects VWF and thrombin-initiated GPIb signaling, and demonstrate the consequent in vivo relevance in the arterial as well as venous circulation during thrombogenesis induced by different models of vascular lesion. We will also develop recent unexpected evidence firmly indicating that mice deficient in GPIb-IX-V function are protected from death in an endotoxin endotoxin-induced model of inflammation, indicating a potentially far-reaching new mechanism that involves GPIb in the regulation of innate immune responses. Altogether, these studies will advance our knowledge of the structural and functional biology of GPIb-IX-V, a key receptor expressed on platelets and, as hypothesized here, on cells of the innate immune system. The new findings resulting from the proposed research will have a significant impact on the understanding, diagnosis and treatment of thromboembolic and inflammatory diseases relevant to human health. 1